XP® Sensitivity Comparison featuring Phospho-GSK-3β #5558

Western blot analysis (Figure 1) demonstrates the superior specificity and sensitivity of #5558.

The concentration of the antibody employed is significantly lower for #5558, further illustrating the sensitivity of the product.

CST #5558

Competitor 1

Competitor 2

Western Blot Dilution

1:1000

1:200

1:500, 1:250

Assay Concentration (µg/mL)

0.29

0.5

1

Recommended Applications

W, IP, IF-IC, F

W, IP

W, IF-IC, F

Species Cross-reactivity

H, M, R, Hm

H, M

H

Figure 1. Jurkat cells (left) were either treated with the PI3 Kinase inhibitor LY294002 #9901, which inhibits GSK-3β phosphorylation, or with Calyculin A #9902, which artificially increases phosphorylation levels by inhibiting phosphatases present in cell extracts. For all antibodies, a signal at the appropriate molecular weight (46 kDa) was detected in the calyculin A-treated sample, however, the signal was strongest with #5558. Note: Competitor 2 also shows a significant number of non-specific bands with greater intensity than the appropriate 46 kDa band. Jurkat cells (right) were either left untreated or treated with the apoptosis-inducing reagent, etoposide, a treatment which is known to reduce phosphorylation of GSK-3β. Neither of the competitor products detect phospho-GSK-3β in either the untreated or the etoposide-treated samples.

XP® Sensitivity Comparison featuring Phospho-S6 #4858

Due to the high level of specificity and sensitivity of #4858, this antibody displays exceptional performance in a broad range of research applications.

CST #4858

Competitor 1

Western Blot Dilution

1:2000

1:1000

Assay Concentration (µg/mL)

0.029

Unknown

Recommended Applications

W, IHC-P, IHC-F, IF-IC, F

W

Species Cross-reactivity

H, M, R, Mk, Sc, (C)

H, (R)

Figure 2. Serial dilutions of extracts from untreated or insulin-treated NIH/3T3 cells (100 nM, 10 min) were detected with #4858 at 1:2000 dilution and the lowest dilution within the manufacturer’s recommended range for the competitor antibody (1:1000). The stronger signal was observed using #4858, with the signal of the competitor antibody barely visible in the lanes with lowest amounts of extract or the untreated lane (which contains basal levels of phospho-S6). Note: The competitor antibody displays cross-reacting bands that are stronger than the band of appropriate molecular weight, showing the lack of specificity of this product.

As shown in Figures 3A and 3B, all three CST antibodies show a single and specific band at the appropriate molecular weight, consistent with CST’s high standard validation requirements and promise of the highest quality.

Quantification of immunofluorescence intensity in a serial dilution on a high content platform revealed greater intensity of #4858 compared to #4856 (Figure 5B).

In a side by side comparison using flow cytometry, greater fluorescence intensity and a greater induction over control at optimal concentration was observed with #4858. Moreover, recommended optimal assay concentration of #4858 is lower than #4856 (Figure 6).

Figure 3A. Western blot analysis of extracts from insulin-treated HeLa cells using serial antibody dilutions of #4858 and #4856, starting at 1 μg/ml. Both antibodies generate a clean and specific signal. However, a much more intense signal was observed with #4858. On the 5 second exposure shown, #4858 generates a strong signal at 1 ng/ml, while the signal from #4856 is much weaker.

Figure 3B. Western blot analysis of a serial dilution of extracts from insulin-treated HeLa cells (100 nM, 10 min) detected with CST selling stocks of #4858, #4856 and #4857 at 1:1000 dilution. Again, the strongest signal was observed using #4858.

Figure 5B. Side by side immunofluorescent titration analysis comparing #4858 with #4856. Optimal concentration for both antibodies was determined to be 0.25 μg/ml. At this concentration signal brightness was 2 fold higher with #4858, demonstrating greater sensitivity.

Figure 6B. Side by side immunofluorescent titration analysis comparing #4858 with #4856. Optimal concentration was determined as 0.25 and 0.5 μg/ml, for #4858 and #4856, respectively. At these concentrations signal brightness was almost 2 fold higher using #4858. Moreover, fold induction over control was also nearly 2 fold higher (not shown). These results indicate greater sensitivity of #4858 by flow cytometry.